Genet. Res., Camb.(1997), 69, pp. 233–236. With 1 figure. Printed in the United Kingdom # 1997 Cambridge University Press 233 SHORT PAPER Random loss of X chromosome at male determination in an aphid, Sitobion near fragariae, detected using an X-linked polymorphic microsatellite marker ALEXANDRA C.C. WILSON*, PAUL SUNNUCKS DINAH F. HALES School of Biological Sciences, Macquarie Uniersity NSW 2109, Australia (Receied 28 October 1996 and in reised form 7 February 1997) Summary This paper reports the first direct molecular evidence that X chromosome loss during determination of male aphids (XO) is random. Clonal and sexual females, and males, of the species Sitobion near fragariae were screened using three polymorphic microsatellite loci. Two loci, Sm10 and Sm17, showed the same heterozygous genotypes in all three aphid morphs. The third, Sm11, was heterozygous for the same two alleles in clonal and sexual females, but of the 25 males screened 11 showed the ‘160’ allele and 14 showed the ‘156’ allele. This result indicates X- linkage of locus Sm11, with random loss of the X chromosome during the formation of male embryos. The possible implications of this result are discussed with respect to aphid sex determination, recombination and chromosome evolution. The Coccoidea (scale insects, mealybugs) constitute 1. Introduction the sister group to the aphids within the Suborder Reproduction in Aphididae encompasses a large range Sternorrhyncha of the Order Hemiptera (Moran et of strategies, including obligate apomictic partheno- al., 1994). There is a wide range of bizarre cytological genesis, and a facultative yearly sexual generation events in different coccoid families, often including a with one to many intervening parthenogenetic genera- form of imprinting of the paternal chromosomes such tions (for review see Hales et al., 1997). Sex de- that they are inactive. For example, in lecanoid scale termination in most aphids is XX}XO. All aphid insects, the paternal chromosomes become hetero- zygotes are originally XX female, but some become chromatic in male embryos at the blastoderm stage, male on receiving only one X chromosome during and are ultimately eliminated during spermatogenesis division of an oocyte in a parthenogenetic female at the second meiotic division, so that males pass on (Orlando, 1974; Blackman & Hales, 1986). Multiple only those chromosomes received from their mothers. X chromosome systems are also known in aphids In diaspidid scale insects, the whole paternal set of (Blackman, 1995). There are still many unanswered chromosomes is completely eliminated in male em- questions about chromosomal behaviour during male bryos at late cleavage (White, 1973). meiosis and male determination in aphids (see above Crema (1981) found an approximately equal ratio references). Male meiosis in aphids is peculiar in that of aborted eggs to viable male eggs in Megoura iciae only those autosomes that associate with an X and suggested that it was attributable to a recessive chromosome survive and the set of autosomes lacking lethal gene on one of the X chromosomes. Further, an X chromosome receives little cytoplasm and does Smith & MacKay (1989) inferred that the ratio of 1:1 not form a viable spermatocyte (for review of early winged to wingless phenotype in males of Acyrtho- work see Blackman, 1987). Factors determining which siphon pisum resulted from a sex-linked gene. Their autosomes become associated with an X chromosome data suggested that X chromosome elimination was are not known. Even during parthenogenetic re- random with respect to chromosomal identity. How- production, in which oogenesis is essentially mitotic, ever, wing phenotype is a highly labile feature in observations of chromosome behaviour suggest the aphids, responding to numerous environmental vari- possibility of X chromosome recombination (Black- ables acting in late embryonic or early larval de- man & Hales, 1986; Blackman & Spence, 1996), but velopment (reviewed by Hille Ris Lambers, 1966; this has not been tested with genetic data. Lees, 1966; Hales, 1976; Kawada, 1987), and it is thus desirable to test for equality of elimination of each X * Corresponding author. Tel: (612) 9850 8223. Fax: (612) 9850 8245. e-mail: awilson!rna.bio.mq.edu.au. chromosome using appropriate molecular markers. Downloaded from https://www.cambridge.org/core. IP address: 170.106.35.93, on 30 Sep 2021 at 06:28:08, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0016672397002747 A. C. C. Wilson et al. 234 It cannot be assumed that there is an equal 2. Materials and methods probability of elimination of either of the X chromo- somes at male determination in aphids, nor of the A single clone (clone 17) of Sitobion near fragariae elimination of either set of autosomes at male meiosis. originating from Tasmania was raised on barley We have previously attempted to address the former seedlings in pots in a temperature-controlled room at issue using allozymes, but did not find a polymorphic 15 mC and an 8 h light}16 h dark photoperiod. Under sex-linked marker in any species studied (D. F. Hales, these conditions parthenogenetic winged and wingless unpublished data). The discovery of an X-linked females, sexual females and males were produced. microsatellite has provided us with a molecular marker Sexual females and males were stored in 70% ethanol. of suitable resolution to study the probability of (Under the same conditions, clones of S. miscanthi, elimination of X chromosomes in aphids at male another aphid of grasses and cereals in Australia, did determination. not produce sexual forms.) Microsatellites are co-dominant, single-locus DNA from individual aphids was extracted using a markers which exhibit a large range of genetic diversity ‘salting-out’ protocol (Sunnucks & Hales, 1996) and and are relatively neutral to selection (Bruford & dissolved in 20 µl of TE Buffer (1 m EDTA, 10m Wayne, 1993; Hughes & Queller, 1993; Estoup et al., Tris base pH 7±5). Microsatellites were cloned and 1995; England et al., 1996). They provide powerful developed from Sitobion miscanthi as described in tools for linkage and population studies. X-linked Sunnucks et al.(1996). The sequence of primers for microsatellites have previously been reported in other loci Sm10 and Sm17 will be published elsewhere, but insects including Drosophila melanogaster (Goldstein in the meantime may be requested from the authors. & Clark, 1995; England et al., 1996) and a mosquito, The sequences of primers to amplify locus Sm11 were Anopheles gambiae (Lanzaro et al., 1995). (5« to 3«): Sm11F aac cct acg ggt aac gcc (®Tm ¯ Sitobion near fragariae is a grass aphid thought to 59±8 mC) and Sm11R ggt acc cct atg tta tta cgc g have arrived in Australia within the last 50 years, and (®Tm ¯ 59±7 mC). is now common in south-eastern Australia. It is Microsatellite polymerase chain reaction (PCR) morphologically similar, but not identical, to S. was carried out in 10 µl reaction volumes with 0±1 µl #+ fragariae (Walker) from the Holarctic region. Mol- of Taq polymerase (0±5 units, Promega), Mg -free ecular evidence also supports a close relationship of reaction buffer, 2 m MgCl#, 200 µ dGTP, dCTP this species with S. fragariae (Sunnucks & Hales, and dTTP, 20 µ dATP, 5–10 pmoles of primers, 5– 1996). 50 ng DNA (typically 1 µl as prepared above) and $$ Males, but not sexual females, of S. near fragariae 0±05 µlof[αP]dATP (10 mCi}ml; Bresatec). The have been reported in field collections in Australia (M. PCR cycling conditions for locus Sm11 were: 94 mC for Carver, personal communication). These aphids under 3 min, four cycles of ‘touch-down’ (annealing first at field conditions appear to be behaving as obligate 55 mC for 30 s and decreasing for the next three cycles parthenogens: we have used microsatellites to dem- (53 mC, 51 mC, 49 mC), extending at 72 mC for 45 s, onstrate the absence of genetic recombination in over denaturing at 94 mC for 15 s) and then cycling for 29 150 Australian S. near fragariae from diverse col- cycles, annealing at 47 mC, with a final extension at lections (Sunnucks et al., 1996, and unpublished 72 mC for 2 min. For Sm10 and Sm17, all annealing data). However, under laboratory conditions we have temperatures were increased by 8 mC. Four microlitres induced a clone of S. near fragariae to produce males of each reaction were run on a 6% acrylamide and sexual females (present study). sequencing gel using the positive control M13 DNA The cloning of microsatellites has given us the tools of the Sequenase version 2.0 kit (United States to investigate X-linkage and, further, we have analysed Biochemicals) as a marker ladder for the precise sizing sufficient males to determine whether the elimination of alleles. Reference DNA can be obtained from the of the X chromosome is random at male deter- authors where confirmation of allelic length identity is mination. We are not aware of previous reports of X- necessary. linked microsatellites (or indeed of any highly variable Lengths of chromosomes in air-dried preparations X-linked single loci) in aphids. The relevance of our (Hales & Lardner, 1988) from Sitobion clones were study to recently published work concerning rDNA measured from photographic prints using vernier (Blackman & Spence, 1996), and the observations of calipers, and X chromosome length as a percentage of ‘aborted’ embryos during the transition to male haploid genome length in each chromosome set was production (Crema, 1981; Searle & Mittler, 1981)is calculated (n ¯ 8; 3 S. near fragariae and 5 S. discussed. We show that microsatellite markers should miscanthi). be valuable in elucidation of cytogenetic observations The G-test for goodness of fit (Sokal & Rohlf, 1995) of aphid male meiosis, and of possible recombination was used to test the observed allelic frequencies in between X chromosomes during parthenogenetic and males at locus Sm11 against equal hemizygous sexual reproduction.